Project Summary/Abstract Background: There is a considerable amount of evidence documenting the importance of ubiquitin conjugation and deconjugation in DNA repair mechanisms, as well as the identity of some the ubiquitin ligases and some of the deubiquitinating enzymes involved. However, the substrates of ubiquitination are still largely controversial and the heterogeneity of the ubiquitin chains involved has not been clearly defined. Recently, unanchored ubiquitin chains have emerged as functional regulators of innate immunity and kinase regulation, however they have not yet been addressed in DNA repair mechanisms. It is important to understand the complete picture of the ubiquitin architecture of DNA repair mechanisms and exploring the functional importance of unanchored ubiquitin chains presents a hitherto overlooked opportunity. Objective: The goal of this proposal is to further characterize the ubiquitin architecture of DNA repair mechanisms in order to be able to exploit them for more successful therapeutic strategies. On the one hand, the possibility of free, unanchored ubiquitin chains will be assessed, as well as the effect of sustained ubiquitin chain formation on DNA repair mechanisms, both of which will underscore the importance of deubiquitinating enzymes (DUBs) as crucial regulators of DNA repair pathways. Specific Aims: (1) Determine the existence and abundance of unanchored ubiquitin chains during DNA damage responses, (2) Determine the effect of sustained ubiquitin chain formation on DNA damage responses, (3) Identify functionally important interacting proteins of alternative ubiquitin-linkages within DNA damage foci. Study Design: At first, polyubiquitin conjugates will be purified from cells treated by DNA damaging agents and ubiquitin conjugates will be assessed for their free ubiquitin chain content. Then, the effect of a conjugation- competent but deconjugation-deficient mutant of ubiquitin that I have recently developed will be tested to initiate a DNA repair response mechanism in the absence of DNA damage. Additionally, the effect of the mutant in making ubiquitin chains of a defined linkage will be tested under DNA damaging conditions to see if sustained ubiquitin chain formation sensitizes cells to DNA damage or inhibits the response. Finally, ubiquitin chain binding proteins will be purified by affinity purification from DNA damage induced cells to identify novel interacting proteins of alternative ubiquitin chains involved in DNA damage by mass spectrometry.